Project #1: Microassisted Fertilization and Genetic Defects - This proposal seeks continued support to determine the molecular basis of non-obstructive azoospermia (no sperm in the ejaculate) in the human. During our current funding period we tested the hypothesis that genomic instability is associated with azoospermia or severe oligospermia (few sperm in the ejaculate). We defined defects in DNA mismatch repair in men with Sertoli cell only syndrome (no spermatogenic cells in the testis, except for occasional rare foci). Loss of MLH1 or MSH2 mismatch repair gene expression in the Sertoli cell only fathers results in a mutator phenotype" with a greatly diminished fidelity of the genome. Absent or abnormal expression of the mismatch repair proteins, MLH1 and MSH2 in Sertoli cell only patients was associated with a high level of microstatellite or genomic instability and a high rate of mutation. Microsatellite instability was associated with expansion of repetitive sequences of DNA associated with neurodegenerative diseases in the testis/sperm and the somatic tissues of Sertoli cell only men. A 64-fold increase in the incidence of a triplet repeat neurodegenerative disease, Myotonic dystrophy, was observed in men with non-obstructive azoospermia compared to the normal population. Because of the intrinsic defect in DNA repair present in the Sertoli cell only father, children conceived by injection of rare sperm (obtained by testicular sperm extraction used in combination with Intracytoplasmic sperm injection [TESE-ICSI]) may be at high risk of developing adult onset neurodegenerative diseases or other gene mutations. The proposed studies focus on the safety of TESE-ICSI for these children and examine whether they exhibit disease range nucleotide repeat expansions. The molecular basis for this instability will be determined (mutation or epimutation in MSH2 and MLH1). We hypothesize that the genomic instability seen differs from that seen in the triplet repeat diseases. We will characterize the paternal transmission by TESE-ICSI of neurodegenerative disease loci alleles. Finally, we will expand our analysis to test the hypothesis that defects in MSH3, 4, and 5 (genes involved in synapsis and crossing over in meiosis, rather than mismatch repair) are associated an increased level of chromosome nondisjunction causing a meiotic arrest and non-obstructive azoospermia. The long term goal of this study is to define the molecular causes of non-obstructive azoospermia and the safety for the offspring when TESE-ICSI is used to achieve an otherwise impossible pregnancy.